Resonance Raman characterization of biotin sulfoxide reductase - Comparingoxomolybdenum enzymes in the Me2SO reductase family

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sp haeroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of O-18/O-16 labeling studies involving water and the alternative sub strate dimethyl sulfoxide and the close correspondence to the resonance Ram an spectra previously reported for dimethyl sulfoxide reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M . K. (1997) J. Am. Chem. Soc. 119, 12906-12916), vibrational modes associat ed with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mon o-oxo-Mo(VI) and des-oxo-Mo(VI) forms with both molybdopterin dithiolene li gands remaining coordinated in both redox states. Direct evidence for an ox ygen atom transfer mechanism is provided by O-18/O-16 labeling studies, whi ch show that the terminal oxo group at the molybdenum center is exchangeabl e with water during redox cycling and originates from the substrate in subs trate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine, How ever, product-induced changes in the Mo=O stretching frequency provide dire ct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate . The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechani sm is proposed.